Visual Cycle Modulation as an Approach
toward Preservation of Retinal Integrity
Claes Bavik1, Susan Hayes Henry1, Yan Zhang1, Kyoko Mitts1, Tim McGinn1, Ewa Budzynski1, Andriy Pashko1, Kuo Lee Lieu1, Sheng Zhong1, Bruce Blumberg2, Vladimir Kuksa1, Mark Orme1, Ian Scott1, Ahmad Fawzi1, Ryo Kubota1*
1Acucela, Inc., 1301 2nd Avenue, Suite 1900, Seattle, Washington 98101, United States of America, 2University of California, Irvine, School of Biological Sciences, 4351 Natural Sciences II, Irvine, California 92697, United States of America
*rkubota@acucela.com
Abstract
Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the im-pact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candi-date in this class of compounds, referred to as visual cycle modulators, is emixustat hydro-chloride (emixustat). Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65), on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activityin vitro(IC50= 4.4 nM) and was found to reduce the production of visual chromophore (11-cisretinal) in wild-type mice following a single oral dose (ED50= 0.18 mg/kg). Measure of drug effect on the retina by electroretinogra-phy revealed a dose-dependent slowing of rod photoreceptor recovery (ED50= 0.21 mg/kg) that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light expo-sure. Pre-treatment with a single dose of emixustat (0.3 mg/kg) provided a ~50% protective ef-fect against light-induced photoreceptor cell loss, while higher doses (1–3 mg/kg) were nearly
100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E) accumulation, chronic (3 month) emixustat treatment markedly reduced lipofuscin auto-fluorescence and reduced A2E levels by ~60% (ED50= 0.47 mg/kg). Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reper-fusion injury produced a ~30% reduction in retinal neovascularization (ED50= 0.46mg/kg). These data demonstrate the ability of emixustat to modulate visual cycle activity and reduce pathology associated with various biochemical and environmental stressors in animal models. Other attributes of emixustat, such as oral bioavailability and target specificity make it an at-tractive candidate for clinical development in the treatment of retinal disease.
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OPEN ACCESS
Citation:Bavik C, Henry SH, Zhang Y, Mitts K, McGinn T, Budzynski E, et al. (2015) Visual Cycle Modulation as an Approach toward Preservation of Retinal Integrity. PLoS ONE 10(5): e0124940. doi:10.1371/journal.pone.0124940
Academic Editor:Stephan C.F. Neuhauss, University Zürich, SWITZERLAND
Received:January 6, 2015
Accepted:March 19, 2015
Published:May 13, 2015
Copyright:© 2015 Bavik et al. This is an open access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability Statement:All relevant data are within the paper and its Supporting Information file.
Introduction
The conversion of dietary vitamin A (all-trans-retinol) into the light-sensitive visual chromo-phore (11-cis-retinal), and its regeneration following photobleaching, is accomplished through a series of enzymatic reactions known as thevisual cycle. Processes of the visual cycle occur within the retinal pigment epithelium (RPE) and in outer segments of rod and cone photore-ceptors (reviewed in [1]). Mobilization of stored vitamin A (all-trans-retinyl esters) is a key step of the visual cycle and is catalyzed by the RPE-specific protein, RPE65. This enzyme per-forms a unique hydrolysis/isomerization reaction to produce the visual chromophore precur-sor, 11-cis-retinol, from retinyl ester stores. Oxidation of 11-cis-retinol yields 11-cis-retinal, which is transferred from apical processes of the RPE to outer segments of rod and cone photo-receptors where it combines with the appropriate opsins to generate light-sensitive rod and cone visual pigments (Fig 1).
Photobleaching of the visual pigments liberates opsin apo-proteins and all-trans-retinal. Under normal physiological conditions, all-trans-retinal is reduced to all-trans-retinol and re-turned to the RPE for recycling. However, under conditions which allow all-trans-retinal to tran-siently accumulate in photoreceptor outer segments (e.g., during intense light exposure or due to a genetic defect), the retina becomes extremely susceptible to oxidative damage. All-trans-retinal is a toxic aldehyde and a potent photosensitizer [2,3]. It can induce lipid peroxidation through the generation of reactive oxygen species (ROS), such as singlet oxygen [4], and cause damage to
Fig 1. Processing of Vitamin A in The Visual Cycle.Enzymatic processing within the visual cycle begins with delivery of vitamin A (all-trans-retinol) from the blood circulation. Upon entry into the RPE, all-trans-retinol is converted to a retinyl ester through the activity of lecithin retinol acyl transferase (1). The resulting all-trans -retinyl ester pool represents a storage form of vitamin A upon which RPE65 acts to generate 11-cis-retinol (2); 11-cis-retinol is then oxidized by an 11-cis-specific retinol dehydrogenase to form the visual
chromophore, 11-cis-retinal (3). The visual chromophore is delivered to rod and cone outer segments (4) where it combines with opsins to form visual pigments (e.g., rhodopsin). Light activation of rhodopsin initiates visual transduction processes and liberates all-trans-retinal as a photoproduct. Reduction of all-trans-retinal, via all-trans-retinal dehydrogenase, produces all-trans-retinol (5), which is transferred back to the RPE for recycling. The continued activity of RPE65 in the light state ensures sustained levels of rhodopsin, closure of ion channels through transducin activation, and reduced oxygen demand.
outer segment proteins [5,6]. Because of its toxic properties, all-trans-retinal is believed to be a key mediator of retinal light damage [7]. Studies demonstrating that depletion of 11-cis-retinal by pharmacologic or genetic means provides protection against light-mediated retinal damage are consistent with this hypothesis [8,9,10].
Because all-trans-retinal is released in a phosphatidylethanolamine- (PE) rich environ-ment, it is prone to a condensation reaction with the primary amine of PE to form a retinal-PE adduct [11]. A similar reaction is thought to occur with free 11-cis-retinal [12]. Retinal-PE adducts may undergo secondary condensation with additional retinal molecules leading to formation of bis-retinoid-PE complexes which can be processed to form stable amphi-philic compounds, such asN-retinylidene-N-retinylethanolamine (A2E) [13,14]. A2E is a well-characterized bisretinoid that has been shown to have several deleterious effects on RPE cells, including generation of ROS [15,16], impairment of lysosomal function [17,18], induc-tion of pro-apoptotic proteins[19], complement activation [20], and upregulation of vascular endothelial growth factor (VEGF) [21]. In animal models of excessive A2E accumulation, compounds that either inhibit visual cycle activity or reduce vitamin A delivery to the RPE are effective to halt the accumulation of A2E and attenuate retinal pathology [22,23,24,25].
While prolonged or intense acute light exposure poses a potential risk for photo-oxidative reti-nal damage, dark adaptation engenders a significant cellular stress associated with high oxygen consumption [26,27]. During dark adaptation, photoreceptors consume large amounts of oxygen to fuel an ion pumping activity that is necessary to maintain electrochemical gradients across the outer segment membrane. In bright light, the channels through which the ions flow are closed, pumping activity ceases and retinal oxygen consumption decreases by 40%–60%, relative to the dark adapted state [28]. The high oxygen consumption of photoreceptors creates an extremely low oxygen tension in the outer retina generating a“sink”for any available oxygen, which can only come from the inner retina. In the context of an emerging, or compromised, inner retina vas-culature, deprivation of retinal oxygen can create a hypoxic environment prompting the aberrant growth of new blood vessels to meet oxygen demands [29,30]. It has been theorized that preven-tion of complete dark adaptapreven-tion, through activapreven-tion of rod phototransducpreven-tion during the dark cycle, may be effective to prevent hypoxia and preserve vasculature of the inner retina [31,32,33].
We have hypothesized that reduction of visual chromophore biosynthesis in the visual cycle could be an effective method to prevent retinal pathology caused by prominent cellular stressors such as light, retinoid byproducts, and oxygen. To evaluate this hypothesis, we have examined the effects of a specific inhibitor of RPE65 in diverse animal models which manifest retinal pa-thology caused by these stressors.
Material and Methods
Emixustat Hydrochloride
Emixustat hydrochloride (emixustat, chemical name: (R)-(+)-3-amino-1-(3-(cyclohexylmethoxy) phenyl)propan-1-ol hydrochloride) is a non-retinoid, small molecule with a molecular weight of 299.84 gram/mole (C16H25NO2•HCl). Chemical synthesis of emixustat is described inS1 Text.
Mice
of ~300 lux for3 days before starting experiments. Mice used in light damage studies were ac-climated under ~300 lux light for20 days. Manipulations during the dark periods and all analy-ses involving retinoids were carried out under dim red light. All procedures were approved by the Acucela Institutional Animal Care and Use Committee and were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Acucela has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) for compliance to policies that promote the humane treatment of animals.
Expression of Visual Cycle Proteins for
In Vitro
Isomerase Assays
cDNAs for humanRPE65(Origene Technologies Inc., Rockville, MD) and lecithin:retinol acyl-transferase (LRAT; American Type Culture Collection, Manassas, VA) were cloned into the pVI-TRO4-neo-mcs vector (Origene Technologies Inc., Rockville, MD), transfected into HEK293H cells, and the stable cell line was further transformed with another expression vector carrying only
RPE65(Origene), to enhance RPE65 expression. Expression was verified by immunoblotting with a mouse anti-RPE65 antibody (Abcam, Cambridge, MA) and by LRAT activity assay. Human cel-lular retinaldehyde-binding protein (CRALBP) cDNA was generated by RT-PCR from total RNA from an ARPE19 cell line (ATCC, Manassus, VA) and expressed in E. coli (pTrcHis2-TOPO TA vector; Invitrogen Corp., Carlsbad, CA). Recombinant CRALBP-His protein was purified using a Ni-Sepharose FPLC system (GE Healthcare Life Sciences, Uppsala, Sweden); purity and quantity were assessed by SDS-PAGE and BCA assay, respectively (Thermo Fisher Scientific Inc., Rock-ford, IL).
In vitro
Isomerase Assays
Assays were performed as described [23], using a homogenate of the HEK293H cell line ex-pressing human RPE65 and LRAT, with 20μM all-trans-retinol (Toronto Research Chemicals;
Toronto, Canada) as substrate. Recombinant human CRALBP (80μg/mL) was added to
facili-tate formation of 11-cis-retinol [23,34]. Reaction mixtures contained 10 mM Bis-Tris propane, pH 7.5, 0.5% BSA, and 1 mM NaPPi. Emixustat (in ethanol, 0.5% v/v) was added to the assay at final concentrations of 10-5to 10-10M; control samples contained the ethanol vehicle. After 1 hour at 37°C, reactions were terminated with methanol, extracted with heptane, dried with a centrifugal evaporator, and resuspended in 150μL of heptane. Retinoids were analyzed by
nor-mal phase HPLC (flow rate = 1.5 mL/min; mobile phase: 0.16% isopropanol, 7.84% ethyl ace-tate, 6% dioxane, and 86% hexane; Agilent 1100 system (Agilent Technologies, Santa Clara, CA), using an Ultrasphere Silica column (5μm, 4.6 mm x 250 mm). Identification of retinoid
peaks was confirmed by on-line spectral analysis (210–450 nm).
In vitro
binding to retinoic acid nuclear receptors
The activity of emixustat (10-5M—10-11M) was evaluated in human retinoic acid receptors (RAR-α, -β, and -γ) and retinoic X receptor-alpha assays performed in triplicate as described27 using 9-cis-retinoic acid, and the synthetic retinoids TTNBP (Biomol Research Labs, Inc., Farmingdale, NY) and AGN203 (kind gift from R. Chandraratna, Allergan Inc., Irvine CA) as positive controls (10-7M—10-11M).
Visual Chromophore Regeneration
white light, 10 min) without anesthesia or mydriatics, returned to the dark for 2 hours, and then euthanized under red light. Enucleated eyes were homogenized in 10 mM Bis-Tris propane, pH 7.3 and 30 mM hydroxylamine, methanol and heptane were added, samples were vortexed and centrifuged (10 min, 13,200 rpm, 4°C). Retinoids in the supernatant were analyzed by normal phase HPLC (flow rate = 1.5 mL/min; mobile phase: 0.15% isopropanol, 14.85% ethyl acetate, and 85% hexane; Agilent 1100 system), with a silica column (Agilent Technologies, 4.6 mm x 250 mm, 5μm). Identification of retinoid peaks was confirmed by on-line spectral analysis (210–450
nm). Retinal oxime levels from emixustat- and vehicle-treated mice were compared to determine the percent chromophore regeneration.
Electroretinography
Recovery of rod photorecptor activity after photobleaching was assessed by electroretinography (ERG) as described [22,35], using the e2Espion ERG system (Diagnosys, Lowell, MA). Dark-adapted (12 hours) BALB/c mice (8 weeks, n = 4/group) were orally dosed with emixustat (0.1–3 mg/kg) or vehicle (water). Mice remained in the dark and ERG measurements were per-formed 4 hours after treatment. Ten minutes before ERG measurements were made, mice were anesthetized with Ketamine:Xylazine (100:10 mg/kg) in PBS (intraperitoneal, i.p., injection; 4μL/g). Left eyes were dilated with 1 drop of 0.5% tropicamide. All measurements were taken
in a dark room under dim red light. Pre-bleach recordings in response to 0.01 cds/m2(n = 5 measurements) were assessed after a 5 minute recovery from red light exposure. Mice were photobleached (400 cd/m2, 30 sec) and scotopic ERG b-wave responses (amplitudes inμV) to
0.01 cds/m2stimuli were recorded every 2 minutes for 50 minutes. Analysis of rod a-wave re-covery was performed in a similar manner using a lower emixustat dose range (up to 0.8 mg/ kg) due to the smaller amplitude of the a-wave, relative to the b-wave.
Light damage
The extent of light damage to the retina after exposure to intense white light was estimated from histological measurement of outer nuclear layer (ONL) thickness as described [36]. After dark adaptation overnight, BALB/c mice (4 mice/group) were treated with a single oral dose of emixustat (0.3–3 mg/kg) or vehicle (water). All groups, except the vehicle-treated dark control group, were treated with 0.5% tropicamide eye drops and subjected to light treatment (8,000 lux of white light for 1 hour) without anesthesia 4 hours post-dose. Mice not exposed to light served as negative (dark) controls. Mice were returned to recover overnight in the dark and ONL thickness was measured after two weeks in normal cyclic light.
Mice were euthanized with CO2, eyes were enucleated and marked to preserve orientation. The eyes were placed in Davidson’s fixative for overnight fixation followed by an overnight wash in 70% ethanol and were then embedded in paraffin. Tissue sections (5μm) were cut
along the vertical meridian of the eye to produce sections of central retina that extended from the superior to the inferior edge. The sections were deparaffinized in xylene and hydrated in an alcohol gradient. The sections were stained with hematoxylin/eosin and mounted. Sections were examined by light microscopy and photography (40 x 10 mag.). ONL measurements for all sections were obtained from comparable regions of the central retina. The number of ONL nuclei intersected by five lines perpendicular to the retina and evenly oriented was counted and the average cell number represented the ONL thickness for that eye.
Accumulation of A2E and Lipofuscin Autofluorescence
Laboratories Inc, Stony Brook, NY). Mice (mixed 129/Sv and C57BL/6 background) were then bred to homozygosity for the Leu450 variant of theRpe65gene, to maximize accumulation of A2E.31Abca4-/-mice 2 months of age were orally dosed for 3 months with 0.03–3 mg/kg/day emixustat (n = 6–8/group) or vehicle (water; n = 4/group). UntreatedAbca4-/-mice (n = 6–8/ group) were evaluated at study onset to provide baseline (Day 0) A2E levels. Mice in the emixu-stat treatment groups remained healthy and active throughout the treatment period. At the end of the dosing period, eyes were enucleated, homogenized in chloroform/methanol and A2E levels were analyzed by HPLC [37]. Histological analyses included four groups of mice: age- and strain-matched wild type mice orally dosed for 3 months with vehicle;Abca4-/-mice orally dosed for 3 months with vehicle;Abca4-/-mice orally dosed for 3 months with 0.3 mg/kg emixustat; and
Abca4-/-mice orally dosed for 3 months with 3.0 mg/kg emixustat. At the end of the dosing peri-od, eyes from these four groups of mice were enucleated, marked to preserve orientation, fixed (4% PFA) and frozen embedded in OCT media. Tissue sections were prepared from comparable regions near the central retina and autofluorescence was imaged using a FITC excitation filter.
Oxygen-induced Retinopathy
The mouse model of oxygen-induced retinopathy (OIR) was used to evaluate effects of emixustat treatment on retinal neovascularization [38]. Seven day-old mouse pups (129/Sv; 10 litters of 5–7 pups per treatment condition) with nursing mothers were subjected to hyperoxia (75% oxygen) for 5 days. On P12, the mice were returned to room air and daily i.p. injections of ruboxistaurin (10 mg/kg, Axon Drugs Pvt. Ltd, India), ruboxistaurin vehicle (5% DMSO), emixustat (0.03–3.0 mg/kg), or emixustat vehicle (water) were administered over the ensuing 5 days. On day 17, the mice were sacrificed, eyes were enucleated and fixed in 4% PFA (2 hours at 4°C) and then trans-ferred to cold PBS. Retinas were dissected from the eyeglobes, dehydrated in cold methanol (15 min) and washed in cold ICC buffer (0.5% BSA, 0.2% Tween20, 0.1% TritonX-100 in PBS). Reti-nas were stained with isolectin B4 in ICC buffer (1μg/μL, overnight at 4°C), followed by a wash
in cold ICC buffer. Four radial cuts were made along the peripheral retina to allow flat mounting. Retinas were prepared in mounting media for microscopic analysis of retinal neovascularization (NV) (Adobe Photoshop).
Analyses
GraphPad Prism software (La Jolla, CA) was used to calculate IC50, EC50, and ED50values; data were fit with sigmoidal-dose response curves (var. slope; 4 param. logistic equations). One-way ANOVA with Tukey multiple comparison tests was used for all statistical analyses.
Results
Inhibition of Isomerase Activity and Visual Chromophore Regeneration
The potency of emixustat to inhibit isomerase activity was evaluated in anin vitroassay system. Homogenates from cells overexpressing RPE65 and LRAT were incubated with all-trans -reti-nol (all-trans-ROL), CRALBP, and increasing concentrations of emixustat. The production
11-cis-retinol (11-cis-ROL) was measured by HPLC (Fig 2A). The data showed a concentration-dependent reduction of 11-cis-ROL production (i.e., inhibition of RPE65 isomerase activity) by emixustat. Analysis of the 11-cis-ROL data gave an IC50value of 4.4 ± 0.59 nM for inhibition of RPE65 isomerase activity (Fig 2B).
a photobleach. Dark-adapted mice were treated with emixustat (0.01–3 mg/kg), photobleached 4 hours after dosing, and returned to darkness for 2 hours to allow regeneration of 11-cis-retinal. The HPLC data showed a dose-dependent reduction of 11-cis- and all-trans-retinal oximes in mice treated with emixustat. The data also showed an accumulation of retinyl esters in emixustat-treated
Fig 2. Inhibition of Isomerase Activity and Visual Chromophore Regeneration.The effect of emixustat on isomerase activity and visual chromophore regeneration was evaluated inin vitroandin vivoassays. Reaction conditions forin vitroassays are described inMethods. Retinoids in the sample extracts were measured by HPLC (RE, retinyl esters; ROL, retinols). Panel A shows representative HPLC chromatographic tracings from a control sample containing no emixustat (black trace) and a sample containing 1μM emixustat (red trace). Note absence of the 11-cis-ROL peak in the sample containing emixustat. The dose-response curve for inhibition of 11-cis-ROL production by emixustat is provided in panel B (each data point represents mean±SEM, n = 8). The mean 11-cis-ROL level in control samples (no emixustat) was taken as 0% inhibition; half maximal inhibitory concentration (IC50) for 11-cis-ROL production in the presence of emixustat is provided in the panel B inset. The effect of emixustat on visual chromophore regenerationin vivowas evaluated in wild-type mice as described inMethods. In these analyses, the amount of 11-cis-retinal production, at 2 hours in darkness following a photobleach (measured by HPLC assyn-andanti-11-cis-retinal oximes,s-11-cis-ROx anda-11-cis-ROx, respectively), in mice treated with varied doses of emixustat was examined. Sample chromatographic tracings of the retinoid profile from a vehicle-treated mouse (black trace) and a mouse treated with 1 mg/kg emixustat (red trace) are shown in panel C (syn-andanti-All-trans-retinal oximes,s-All-trans-ROx anda-All-trans-ROx, and all-trans-retinyl esters, REs, are also shown). Note reduced level ofsyn-andanti-11-cis-retinal oxime peaks and increased REs in the trace from the emixustat-treated mouse. The dose-response curve for inhibition of visual chromophore production by emixustat is provided in panel D (each data point represents mean±SEM, n = 8). The mean 11-cis-retinal oxime level in control samples (no emixustat) was taken as 0% inhibition. The calculated half maximal effective dose (ED50) for inhibition of visual chromophore production by emixustat is provided in the panel D inset.
mice, consistent with reduced mobilization of this storage pool due to RPE65 inhibition (Fig 2C). The ED50for the effect of emixustat on inhibition of 11-cis-retinal oxime production was deter-mined to be 0.18 mg/kg (Fig 2D). Importantly, the effect of emixustat was reversible; 11-cis-retinal oxime levels returned to near control levels by 24 hours after dosing.
Activation of Retinoic Acid Receptors
Although emixustat is not a retinoid and, therefore, should neither bind nor activate retinoic acid receptors, it can affect retinoid levels in the visual cycle. Therefore, we examined the effect of emixustat on RAR and RXR binding/activationin vitro. The positive controls (TTNBP, AGN203) activated RAR and RXR as expected. However, no activation was observed with emixustat at concentrations from 10-5to 10-11M (data not shown).
Pharmacodynamic Effect
The effect of emixustat on the physiological response of the retina to light was assessed by evaluat-ing recovery of rod photoreceptor function by ERG after photobleachevaluat-ing (Fig 3). Dark adapted mice were treated with increasing doses of emixustat (0.1–3 mg/kg) or vehicle; 4 hours after dosing, animals were photobleached (400 cd/m2, 30 sec) and recovery of rod function was recorded using scotopic stimuli (0.01 cds/m2). Representative ERG traces obtained from mice in each treatment condition are shown inFig 3A. In vehicle-treated mice, b-wave amplitudes were robust and recov-ered to pre-bleach values within 40–50 minutes. Meanwhile, in mice treated with emixustat, b-wave response amplitudes were diminished and recovery was slowed in a dose-dependent manner (Fig 3B). The dose associated with half-maximal suppression of rod b-wave recovery (ED50) was determined to be 0.21 mg/kg. Calculation of the ED50for a-wave recovery gave a value of 0.18 mg/ kg, which was comparable to the b-wave ED50(not shown). Suppression of rod photoreceptor am-plitude recovery induced by emixustat was reversible; between 16 to 24 hours after dosing, the rate of amplitude recovery equaled or exceeded the rate in vehicle-treated mice (data not shown).
Light Damage
Albino (BALB/c) mice were used to assess the effects of emixustat on protection from light damage. Dark adapted, untreated albino mice showed a healthy retinal morphology with ~10 rows of nuclei in the ONL (Fig 4A, Dark Control). Exposure to 8,000 lux white light for 1 hour caused near complete destruction of the ONL in untreated mice (Fig 4A, Light Control). Pre-treatment with a single dose of emixustat provided dose-dependent protection of the ONL (Fig 4A, Emixustat 0.3 mg/kg and 1.0 mg/kg). Measurement of the ONL thickness (number of nu-clei) showed a reduction from 10 nuclei in the Dark Control to 2.5 nuclei in the Light Control (Fig 4B). In contrast, ONL thickness in mice pre-treated with emixustat was well preserved. Mice treated with either 1.0 or 3.0 mg/kg emixustat showed a statistically significant thicker ONL compared to the vehicle treated group (t-test, p<0.01). The calculated ED50for preserva-tion of the ONL in emixustat-treated mice was 0.20 mg/kg.
Accumulation of A2E and Lipofuscin Autofluorescence
due to the accumulation of A2E and related compounds (compare Fig5Aand5B). Treatment ofAbca4-/-mice with emixustat (3 months daily treatment, 0.3 or 3 mg/kg/day) markedly re-duced RPE autofluorescence (Fig5Cand5D, respectively). Analysis of A2E levels in all groups ofAbca4-/-mice (2 month old controls, and 5 month old mice treated with either vehicle or emixustat) revealed a dose-dependent reduction of A2E (Fig 5E). Emixustat doses above 0.03 mg/kg/day produced a statistically significant reduction of A2E compared to vehicle-treated
Abca4-/-mice (p<0.001). The ED50for the effect of emixustat on reducing accumulation of A2E was 0.47 mg/kg/day.
Fig 3. Pharmacodynamic Effect of Emixustat on Rod Function.The recovery of rod photoreceptor function following a photobleach was measured by ERG. Mice were treated with varied doses of emixustat (n = 4/dose group), or vehicle (n = 4/group), and ERG responses were recorded every 2 minutes for 50 minutes using a light stimulus 0.01 cd*s/m2. Panel A shows representative ERG waveforms in vehicle- and emixustat-treated mice. Arrows in panel A indicate the b-wave amplitude peaks which are plotted as a function of recovery time after photobleach in panel B. B-wave response amplitudes (μV), at each
corresponding time point of recovery, are shown as mean values±SEMfor each of the treatment groups
(panel B). Emixustat treatment caused a dose-dependent suppression of b-wave response amplitudes. The dose required for half-maximal suppression of ERG b-wave recovery was determined to be 0.21 mg/kg.
Oxygen-induced Retinopathy
We hypothesized that reduced chromophore biosynthesis might be effective to prevent complete dark adaptation and, thus, may potentially reduce oxygen consumption in the inner retina and preserve integrity of the retinal vasculature. To explore this possibility, we administered emixu-stat (0.03–3 mg/kg) to OIR mice during the period of reperfusion injury (P12—P17) and exam-ined the extent of retinal NV in flat mounted retinas. Ruboxistaurin, a PKCß inhibitor that is known to inhibit VEGF-mediated retinal NV, was included a positive treatment control. Daily administration of emixustat during P12-P17 produced a dose-dependent reduction in retinal NV. The 3 mg/kg/day dose of emixustat reduced the extent of retinal NV by ~30%, which was comparable to reduction observed with a 10 mg/kg ruboxistaurin (Fig 6A). The ED50for reduc-tion of retinal NV in emixustat-treated mice was 0.46 mg/kg. Representative retinal flat mounts from an untreated, normoxic control, an untreated OIR control, and in a 3 mg/kg emixustat-treated mouse are shown in panels B, C, and D, respectively.
Fig 4. Protection from Light Damage.The ability of emixustat to provide protection from light damage was assessed as described inMethods. Mice received a single dose of emixustat, or vehicle, prior to light exposure (8,000 lux white light, 1 hour). Histological analyses and determination of ONL thickness was performed following a 2-week recovery period. Panel A shows tissue sections prepared from untreated, dark-adapted mice, untreated light-exposed mice, and mice pre-treated with either 0.3 or 1.0 mg/kg emixustat (ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer). Quantitative analysis of ONL layer thickness (panel B) was used to calculate the ED50for preservation of the ONL in emixustat-treated mice (ED50= 0.20 mg/ kg). There was a statistically significant preservation of ONL thickness in mice treated with 1.0 or 3.0 mg/kg emixustat compared to the vehicle-treated, light control group (*, p<0.01).
Discussion
The objective of the present study was to evaluate reduction of visual chromophore biosynthe-sis as a means to preserve retinal integrity in animal models of human retinal pathology. We were particularly interested in photooxidative stress, accumulation of retinoid byproducts and retinal hypoxia as these are suspected to be prominent mediators of photoreceptor damage and degeneration in human retinal diseases (e.g., age-related macular degeneration (AMD), Star-gardt disease, proliferative diabetic retinopathy, and diabetic macular edema). Our approach was to develop an oral non-retinoid, small molecule inhibitor of RPE65 (emixustat) and assess treatment effects in mouse models of light-mediated retinal damage, aberrant lipofuscin accu-mulation, and oxygen-induced retinopathy. Biochemical and electrophysiological analyses of visual chromophore biosynthesis demonstrated that emixustat potently inhibits RPE65 in a dose-dependent, reversible manner. In each model studied, emixustat treatment either im-proved the anatomical phenotype or completely preserved photoreceptor integrity. Notably, the dose required to produce 50% of the maximal treatment effect (ED50) in each of the models
Fig 5. Reduced Lipofuscin Autofluorescence and A2E Accumulation.The effect of emixustat on lipofuscin autofluorescence and A2E levels was examined in an animal model of autosomal recessive Stargardt disease (Abca4-/-mice). Histological analysis of lipofuscin autofluorescence in untreated, strain- and age-matched wild-type mice, andAbca4-/-mice treated with either vehicle or emixustat (0.3 or 3.0 mg/kg) is shown in panels A—D, respectively. All mice were 5 months of age. Lipofuscin fluorophores were extracted from RPE eyecups and analyzed by HPLC as described inMethods. Representative chromatograms from eyecup extracts of mice treated with emixustat (3 mg/kg for 3 months) and vehicle (red and black tracings, respectively) are shown in panel E. UV-vis spectra associated with the indicated peaks (numbered 1–5) are shown in the panel inset. Peak 1 in the chromatogram was determined to be A2E based upon spectral identity and co-elution with an authentic A2E standard. Quantitative analysis of A2E levels (based on area units of peak 1) in
Abca4-/-mice is shown in panel F (peaks 2–4 were not quantified). Numbers of mice analyzed for A2E quantitation are as follows: Day 0: n = 17; Vehicle: n = 6; 0.03 mg/kg: n = 6; 0.1 mg/kg: n = 7; 0.3 mg/kg, n = 8 1.0 mg/kg, n = 8; 3 mg/kg, n = 8. A2E levels increased from ~5 to ~20 pmoles/eye over a 3-month period in vehicle-treatedAbca4-/-mice.Abca4-/-mice treated with emixustat showed a dose-dependent reduction of A2E which was statistically significant at doses0.30 mg/kg/day, relative to vehicle treatedAbca4-/-mice (*, p<0.05). The ED50for the effect of emixustat on reducing accumulation of A2E was 0.47
mg/kg/day.
studied was comparable (0.20–0.47 mg/kg) and was similar to the ED50values determined for reduction of visual chromophore biosynthesis (ED50= 0.18 mg/kg) and the pharmacodynamic effect of emixustat (reduction in rod photoreceptor recovery following a photobleach, ED50= 0.21 mg/kg). These relationships suggest that inhibition of RPE65 by emixustat, may be the pri-mary mechanism of action underlying the observed treatment effects.
Fig 6. Reduced Retinal Neovascularization.The effect of emixustat on retinal neovascularization was studied in the mouse OIR model. Seven day-old mouse pups were subjected to hyperoxia (75% oxygen) for 5 days. On P12, the mice were returned to room air and daily treatments with ruboxistaurin (10 mg/kg), emixustat (0.03–3.0 mg/kg), or appropriate vehicles were administered as described inMethods. Retinal flat mounts were prepared and areas of NV were quantified; these data were compared to data from control mice that were maintained in a normoxic environment (21% oxygen). Mice that were moved from a hyperoxic to normoxic environment, without treatment, showed a significant extent of retinal NV (~30% of the retinal area). Treatment with the ruboxistaurin (positive control) reduced the area of NV to ~20% of the total retinal area. In mice treated with emixustat, a dose-dependent reduction in retinal NV was observed. The reduction in NV at the highest emixustat dose (3.0 mg/kg/day) approached a level that was comparable to that obtained with ruboxistaurin (Fig 6A;*, t-test, p<0.05). The ED50for reduction of retinal NV in emixustat-treated mice was 0.46 mg/kg/day. Representative retinal flat mounts from an untreated, normoxic control, an untreated OIR control, and in a 3 mg/kg emixustat-treated mouse are shown in panels B, C, and D, respectively. Areas of NV, outlined in red tracings, were identified and quantified using Adobe Photoshop software.
Inhibition of visual cycle function as a potential therapeutic approach for the treatment of reti-nal disease, or preservation of retireti-nal function, has been an active area of study during the past sev-eral years. Previous investigations have focused primarily on reduction of lipofuscin fluorophores and/or protection from light damage as pre-clinical efficacy measurements using retinoid-based compounds [4,7,9,25,40]. Of particular note are the systematic studies of retinylamine (Ret-NH2) conducted by Palczewski and coworkers [4,9,40]. Originally developed as a transition state analog for the RPE65 isomerization reaction, Ret-NH2was shown to be a potent inhibitor of RPE65 [22,23]. The therapeutic potential of Ret-NH2was examined in a line of mice which rapidly accu-mulated all-trans-retinal condensation products due to a null mutation in theAbca4and retinol dehydrogenase 8 (Rdh8) genes. The aberrant phenotype of these mice, which included early accu-mulation of lipofuscin/A2E, drusen, basal laminar deposits, Bruch's membrane thickening, and choroidal neovascularization, was significantly reduced following treatment with Ret-NH2[4,40]. These findings provided additional evidence for the link between accumulation of retinaldehyde toxins and RPE/photoreceptor degeneration and demonstrated that pharmacologic intervention in the visual cycle could be a tractable therapeutic approach.
Emixustat possesses important pharmacologic attributes which distinguish it from other small molecule RPE65 inhibitors. First, emixustat is a non-retinoid compound. As such, it cannot be metabolized to form a retinoid and is shown in the present study to have no activity against retinoic acid receptors. Secondly, emixustat demonstrates an extremely high and selec-tive inhibitory potency against RPE65 isomerase activity (IC50= 4.4 nM), providing the po-tential for less drug exposure in chronic dosing regimens. Finally, we have observed an anti-angiogenic effect of emixustat which has not been previously demonstrated with other RPE65 inhibitors, but is explainable based upon the mechanism of emixustat action.
High oxygen consumption of photoreceptors during dark adaptation is believed to produce ischemia within the inner retina promoting aberrant new vessel growth. In the light state, activa-tion of phototransducactiva-tion significantly reduces oxygen demand of photoreceptors. Meta-II-mediated activation of transducin is key in this process. Importantly, apo-opsin is also capable of activating transducin and can produce an adaptation state much like that produced by real light [41,42,43]. Treatment with emixustat specifically reduces visual chromophore levels with-out affecting levels of the opsin protein. Thus, it is conceivable that during emixustat treatment, apo-opsin could accumulate in amounts sufficient to stimulate phototransduction which would decrease oxygen demand during the dark state and alleviate inner retinal ischemia. This effect would explain reduced retinal NV in OIR mice treated with emixustat.
In summary, data from the present study provide strong evidence that reduction of visual chromophore biosynthesis is an effective method to alleviate photoreceptor cell stress and pre-serve retinal integrity. The ability of emixustat to specifically target RPE65 and attenuate retinal pathology elicited by three prominent and distinct cellular stressors provides an impetus to ex-plore therapeutic approaches in the treatment of degenerative retinal diseases in which these stressors have been implicated.
Supporting Information
S1 Text. Synthesis of Emixustat Hydrochloride. (DOCX)
Acknowledgments
Todd Haight, Olena Iatsenko, David Leung and Lance Lindlief of Acucela Inc. for discussions and technical contributions.
Author Contributions
Conceived and designed the experiments: CB SHH EB SZ BB VK MO IS AF. Performed the ex-periments: SHH YZ KM TM EB AP KLL SZ BB VK. Analyzed the data: CB SHH YZ KM TM EB AP KLL SZ BB VK MO IS AF RK. Contributed reagents/materials/analysis tools: CB BB VK MO IS. Wrote the paper: SHH CB MO.
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